Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.
Carbazole 1,9a-二氧化酶(CARDO)由末端氧化酶(CARDO-O)和电子传递组件组成。CARDO能够对多种底物催化特定的氧化反应:对
喹啉和二苯并-对-二氧的角氧化,对
蒽的侧氧化,以及对弗鲁安的亚甲基碳和
二苯并噻吩的
硫的单氧化。为了阐明决定其独特底物特异性的分子机制,生成并表征了17个CARDO-O的定点突变体,这些突变体位于
氨基酸残基I262、F275、Q282和
F329,这些残基通过CARDO-O的晶体结构形成围绕
铁活性位点的底物相互作用壁。
F329的替换显著降低了氧化活性。然而,几个突变体与野生型酶相比,产生了不同的产物:I262V和Q282Y(1-羟基
喹啉),F275W(
4-羟基弗鲁安),F275A(未鉴定的弗鲁安烯顺式二氢二醇),以及I262A和I262W(单羟基
二苯并噻吩)。这些结果表明,不同底物可能以不同于野生型酶的取向结合在CARDO-O突变体的活性位点。